Histidine decarboxylase (Hdc) is responsible for histamine formation in living organisms. Although histamine produces many physiological effects in animals (e.g. vasodilation, hypotension, HC1 secretion and gastric ulcer formation, anaphylaxis, allergic effects, etc.), Hdc has been studied in pure form only recently. Studies have shown that two classes of Hdc enzymes exist. One class, present in Gram-positive bacteria, (e.g. Lactobacillus sp.) has a catalytically-essential pyruvoyl residue at the active site and no pyridoxal 5'-phosphate (PLP). In the other class, found in Gram-negative bacteria (e.g. Morganella AM-15) and animals, PLP is the essential cofactor. Amino acid sequences of a single Hdc from each class are known; they are not homologous and the quaternary structures are very different. Our specific goals, all of which are aimed at determining the detailed reaction mechanisms of these enzymes, are: (1) Obtain expression of the hdc gene from Morganella in E. coli and develop procedures for purification of the expressed protein in convenient amounts for covalent modification procedures and for crystallization. If the latter is achieved, a collaborating crystallography group will study the 3-dimensional structure of the enzyme. (2) If (1) is successful, then, by use of synthetic mismatched probes and cloning techniques to obtain site-specific mutations in this hdc gene, isolate the resulting expressed Hdc's, and determine their properties to assist in judging the essentiality of specific amino acid residues that have been implicated in enzyme action by other data. These include lysine 232, which forms the azomethine link to PLP in the native enzyme; histidine 231, which precedes lysine in the PLP-binding site of Hdc, arginine decarboxylase, lysine decarboxylase and glutamic acid decarboxylase; serine 229, which also is invariant in the PLP-binding site of these enzymes; and serine 322, which is destroyed when the suicide inhibitor, yield-fluoromethylhistidine, acts on Hdc. Crystals of such modified enzymes, if obtained, will also be subjected to x-ray examination. (3) We will clone and sequence hdc genes from Clostridium perfringens and from Klebsiella pneumoniae. The encoded protein sequences will provide an independent approach to determining amino acid residues that must be conserved for activity of Class I and Class II Hdc's.